Molecular diagnostics

Molecular diagnostics is a family of techniques for the analysis of biomarkers in the genome and proteome, and how their cells express their genes as proteins, applying molecular biology to medical testing.In medicine, the technology is used to diagnose and monitor disease, detect risk, and decide which therapy is most effective for an individual patient, Introduction In agricultural biosecurity, akin to monitoring crop and livestock disease, estimating risk and decide what isolation measures must be taken.By analyzing the specifics of a patient and their disease, molecular diagnostics offer the promise of personalized medicine.These tests are useful in a range of medical specialties, including infectious disease, oncology, human leukocyte antigen typing (to study and predict immune function), coagulation, and pharmacogenomics the genetic prediction of which drugs will be most effective.: v -vii They overlap with clinical chemistry (humoral medicine tests).

Techniques

Development from research tools

The industrialization of molecular biology assays has made their use in the clinic practical.Preface Miniaturization into a single handheld device can bring medical diagnosis into the clinic, office or home.Introduction Clinical laboratories require high standards of reliability; diagnostics may require certification or be subject to medical device regulations.As of 2011, some U.S. clinical laboratories still use test kits sold for "research use only." Laboratory processes need to comply with regulations such as the Clinical Laboratory Improvement Amendments, Health Insurance Portability and Accountability Act, Good Laboratory Practices, and Food and Drug Administration regulations in the United States.Laboratory information management systems help by keeping track of these processes. Regulations apply to employees and supplies. As of 2012, 12 U.S. states require molecular pathologists to be licensed; several boards, including the American Board of Medical Genetics and the American Board of Pathology, certify technicians, supervisors, and laboratory directors.

Automation and sample barcoding maximize throughput and reduce the potential for error or contamination during manual handling and reporting of results. A single device that performs inspection from start to finish is now possible.

Assays 

Molecular diagnostics use in vitro bioassays such as PCR-ELISA or fluorescence in situ hybridization.The assay detects a molecule (usually in low concentration) that is a marker of disease or risk in a sample taken from a patient.Preserving samples prior to analysis is critical. Manual handling should be minimized.Fragile RNA molecules present certain challenges.It provides a measure of gene expression as part of the cellular process of expressing genes as proteins,but is easily hydrolyzed and broken down by the ever-present RNAse enzymes. Samples can be snap frozen in liquid nitrogen or incubated in preservatives.Because molecular diagnostic methods can detect sensitive markers, these tests are less invasive than traditional biopsies.For example, a simple blood sample is sufficient to extract genetic information from tumors, transplants or unborn fetuses due to the presence of cell-free nucleic acids in human plasma.ch 45 Many (but not all) molecular diagnostic methods are based on nucleic acid detection using the polymerase chain reaction (PCR) to greatly increase the number of nucleic acid molecules, thereby amplifying the target sequence in a patient sample. Introduction PCR is a method of amplification using template DNA to synthesize primers, DNA polymerase, and dNTPs.The mixture is cycled between at least 2 temperatures: a high temperature for denaturing double-stranded DNA into single-stranded molecules and a low temperature for primers to hybridize to the template and a polymerase to extend the primers.Each temperature cycle theoretically doubles the number of target sequences.Detection of sequence variations using PCR typically involves the design and use oligonucleotide reagents Amplifies the variant of interest more efficiently than the wild-type sequence.PCR is currently the most widely used method of DNA sequence detection.Detection of markers may use real-time PCR, direct sequencing: ch 17 microarray - prefabricated chip that tests many markers at once, ch 24 or MALDI-TOF.The same principle applies in the proteome and genome. High-throughput protein arrays can use complementary DNA or antibody conjugation, so many different proteins can be detected in parallel.Molecular diagnostic tests vary widely in sensitivity, turnaround time, cost, coverage, and regulatory approval.They also differ in the level of validation applied in the laboratories where they are used.Therefore, robust local validation and use of appropriate controls in accordance with regulatory requirements is required, especially if the results can be used to inform patient treatment decisions.